Triclosan is a broad spectrum antimicrobial agent that functions by inhibiting fatty acid biosynthesis. It is incorporated into hospital and household antiseptic products alike. Previous research unveiled two separate mechanisms of bacterial resistance to the antimicrobial agent -- modification of the drug site of action and active efflux from exposed cells. Both mechanisms, however, conferred resistance to triclosan concentrations lower than those used in antiseptic formulations. The PI has recently isolated three different microorganisms, including a fluorescent Pseudomonas sp. strain called TriR, that are resistant to triclosan at concentrations equivalent to those of antiseptic formulations. The primary research objective is to determine the resistance mechanism of these organisms, which is hypothesized to function through degradation of triclosan. When plated on triclosan-containing media, these organisms produce a clearing zone around the colonies. High pressure liquid chromatography will be utilized to determine if the triclosan concentration decreases over time in these cultures. It is also important to determine if triclosan resistance is plasmid-borne and therefore transferable. This will be accomplished through plasmid curing and triparental mating experiments between the resistant organisms and susceptible bacterial such as Escherichia coli. Finally the gene responsible for triclosan resistance in Pseudomonas sp. TriR will be isolated. This will be accomplished by transforming susceptible E. coli with a Pseudomonas sp. TriR cosmid library with subsequent selection on triclosan-containing medium. Since it is unlikely that a modification of fatty acid biosynthesis or active efflux of triclosan would result in the clearing of the suspended antibiotic from bacterial growth media, the PI expects to uncover a new triclosan resistance mechanism. The resistance determinant isolated from TriR will then be sought in the other isolates through hybridization analysis. The results of these proposed will provide valuable information on a novel mechanism of resistance to the antimicrobial agent triclosan at commercially significant concentrations and will provide a basis for future molecular and biochemical studies.